| Background and ObjectiveCell apoptosis is also called programmed cell death,it is a active programmed physiological process. Cathepsin B(Cathepsin B,CB)involved in the initial part of apoptosis and it plays an important role in cerebral ischemia and reperfusion neuronal apoptosis.It is currently relatively rare ischemia-reperfusion model in the Cathepsin-B-mediated apoptosis pathway. pMkk6 is an important MAP kinase kinase that function as upstream activater of P38 MAPK by directly phosphorylating the dual-site Thr-Gly-Tyr in P38 kinases. Eukaryotic cell through this important signal transduction to mediated the extracellular signal to the intracellular, involve the stress response in the body, play a important regulatory function. In order to investigate the role of CB in cerebral ischemia injury and the molecular mechanism of CB in lysosome pathway of cell apoptosis,we explore the correlation between CB and mkk6 which was important upstream actin in P38-MAPK signaling pathway in cerebral ischemia injury. and used the application of fasudil,a inhibitor of Rho kinase,to observe its influence of CB and pMkk6 and the lysosomal neuronal apoptosis as well as its neuroprotective effects.for clinical treatment of cerebral ischemia and thus provide a theoretical basis and treatment ideas.Methods141 healthy male Sprague-Dawley rats weighing 250-300g were randomly divided into four groups: sham-operated control group (n=11), cerebral ischemia model group (n=65) and fasudil-treated group (n=65). Temporary middle cerebral artery occlusion (MCAO) model was applied.Reperfusion at the time of 2 hours after ischemia.Immediately, rats received intraperitoneal injections of 8mg/kg of Fassudil for the Fassudil -treated group and same volume physiological saline for the other groups,and inject q12h after reperfusion. Then, neurolgical behavior evaluation were evaluated by the methods of Longa's scoring. Rats were sacrificed respectively at 2h, 6h, 12h, 24h, 48h after reperfusion. The cerebal infarction volume were evaluated by the method of TTC staining.The expression of CB and pMkk6 protein were measured by the method of Immunofluorescence. The neuronal apoptosis was detected by the method of terminal deoxynucleotidy1 transferase-mediated dUTP-biotin nick end labeling(TUNEL).Activation of lysosome in neurons in ischemic cortex was observed using electron microscopy..Results1. 76.2%rats were induced ischemic neurological deficits.Compared with the rats of model group,fasudil could reduce neurological deficits significantly beginning at 24h after reperfusion(P<0.05).2. TTC staining showed:normal and sham-operated rats were no infarction and also The TUNEL positive cells were hardly found.The cerebral infarction volume and apoptosis cell counting were increasing gradually with the advance of time after cerebral ischemia-reperfusion and reach a peak at 48h after reperfusion.Compared with the rats of model group,fasudil could reduce those damage(P<0.05)3. A certain amount of CB and pMkk6 protein was detected in the cerebral cortex of sham and normal rats.The expression of CB and pMkk6 protein in the model group were present dynamic changed.The expression level of CB and pMkk6 protein all increased significantly at 2h after reperfusion,peaked at 24h and12h.The changes of the fasudil-treated group was similar with the model group,but the amount reduced remarkably(P<0.05).4. Electron microscopy showed early stage of ischemic penumbra , the volume and the number of lysosome were increased and changes to the secondary lysosome, the electromicroscopy showed nerve cell apoptosis or necrosis later postischemia. Conclusions1. The upregulation of the protein CB after cerebral ischemia and reperfusion.may be involved in cell apoptosis through lysosome pathway,the upregulation of the protein pMkk6 after cerebral ischemia and reperfusion.may be involved in cell apoptosis through pathophysiology of nerve Ischemic injury.2. The inhibitor of Rho kinase fasudil may play an anti-apoptosis role through stabilizing the lysosomal membrane and reduce the release of CB protein.Fasudil which the inhibitor of Rho kinase could inhibit the phosphorylation and expression of which was the critical upstream kinase pMkk6 protein in apoptotic P38/MAPK signal transduction pathway, and inhibit neuronal apoptosis and play a protective role in neurons reduce neurological deficits and cell apoptosis and after cerebral ischemia reperfusion in rats. |
PDF Full Text Request